Interpretive Summary: House flies live and breed in septic environments, and therefore have been implicated in the dissemination and transmission of bacterial diseases affecting humans and livestock. Some previous studies have used either molecular (PCR) or culture methods to detect pathogens associated with flies, but likely have underestimated the presence of many microbes, especially those that are in low abundance, unculturable, or undetectable by standard molecular methods. The innovative approach of this study was to use, and compare, both molecular (culture-independent) and bacteriological (culture-dependent) techniques to detect strains of bacteria in house flies and thereby more accurately assess the diversity of microbiota in these insects. By sequencing a culture-independent library of PCR products, additional species not detected by culture were revealed, which helps in improving the detection of microbes in flies. Some species that were detected are important pathogens of humans and other animals. This provides further evidence that house flies serve as reservoirs, and possible vectors, of diverse bacteria that are of medical and veterinary importance.

Technical Abstract:
House flies (Musca domestica L.) are cosmopolitan, ubiquitous, synanthropic insects that serve as mechanical or biological vectors for various microorganisms. To fully assess the role of house flies in the epidemiology of human diseases, it is essential to understand the diversity of microbiota harbored by natural fly populations. This study aimed to identify the diversity of house fly gut bacteria by both culture-dependent and culture-independent approaches. A total of 102 bacterial strains were isolated from the gut of 65 house flies collected from various public places including a garden, public park, garbage/dump area, public toilet, hospital, restaurant/canteen, mutton shop/market, and house/human habitation. Molecular phylogenetic analyses placed these isolates into 22 different genera. The majority of bacteria identified were known potential pathogens of the genera Klebsiella, Aeromonas, Shigella, Morganella, Providencia, and Staphylococcus. Culture-independent methods involved the construction of a 16S rRNA gene clone library, and sequence analyses supported culture recovery results. However, additional bacterial taxa not determined via culture recovery were revealed using this methodology and included members of the classes Alphaproteobacteria, Deltaproteobacteria, and the phylum Bacteroidetes. Here, we show that the house fly gut is an environmental reservoir for a vast number of bacterial species, which may have impacts on vector potential and pathogen transmission.